Gilteritinib

Human chronic myeloid leukemia (CML) in blast crisis cell line (K562) was procured from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37°C, 5% CO2 in complete medium, containing Iscove’s Modified Dulbecco’s Medium (IMDM), 1X penicillin/streptomycin (Gibco), and supplemented with 10% (v/v) fetal bovine serum (FBS).
To differentiate K562 into Mk, cells were treated with Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA), gilteritinib, quizartinib, dubermatinib (TP-0903), and midostaurin for 48–72 h as previously described (15 (link)). Where indicated, low-serum medium supplemented with 1% FBS and 1 μM imatinib were used.
gilteritinib, quizartinib, dubermatinib (TP-0903), and midostaurin were from Cayman chemical (Ann Arbor, MI, USA). All small molecules were dissolved in DMSO and store in −20°C. pMSCV-Flt3-ITD-Y591F/Y919F was procured from Addgene (Addgene.org). Flt3-ITD-Y591F/Y919F cassette was subcloned into lentiviral vector to produce viral particles using 293T cell.

Cell culture medium (RPMI 1640, Iscove’s Modified Dulbecco’s Medium—IMDM) was purchased from Fisher Scientific. PI3K and CDK6 inhibitors (idelalisinb, duvelisib, alpelisib, copanlisib and palbociclib) were also purchased from Fisher Scientific. Quizartinib was obtained from AdipoGen Life Sciences. Gilteritinib was purchased from Cayman Chemical Company. Abemaciclib was purchased from Selleck Chemicals. Each inhibitor was dissolved in dimethyl sulfoxide (DMSO). Pierce Protease Inhibitor Mini Tablets were purchased from Fisher Scientific. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega.